The controlled delivery of antibodies into cells is currently of great commercial and scientific interest. It was once thought that intact antibody molecules (both endogenous and exogenous) were not able to penetrate viable cells. However, there is now much research that indicates otherwise. Mature autoantibodies penetrating living cells are thought to participate in the pathogenesis of diverse autoimmune diseases, through inducing apoptosis of healthy tissues and cells. The antibodies may also contribute to the breakdown of self-tolerance through presentation of self-antigens to the immune system. The penetration of naturally occurring autoantibodies into immature lymphoid cells may have a physiological role in the immune repertoire in healthy individuals. Increasing interest is being paid to the potential immunotherapeutic role of penetrating antibodies as tools to deliver drugs, isotopes or genes into cells (Ruiz-Arguelles, A., et al, Antibody penetration into living cells: pathogenic, preventive and immuno-therapeutic implications, Current Pharm Design, 2003, 9, 1881). A non-toxic delivery system for intracellular delivery of inherently non-penetrating antibodies could therefore be of great utility.
The delivery of antibodies into cells can be a particular problem either where large numbers of cells are to be analysed or where one wants to study adhesive as well as non-adhesive cells. Several approaches to introduce proteins and other components into cells have been published, such as electroporation, scrape loading, or delivery via liposomes.
Some have reported on other approaches, such as the use of a combination of a novel IgG-capturing protein and hemaglutinating virus of Japan envelope (HVJ-E), an inactivated Sendai virus particle, which can deliver a variety of molecules into mammalian cells via membrane-fusing activity (Kondo, Y. et al, Efficient delivery of antibody into living cells using a novel HVJ envelope vector system, J Immunol Methods, 2008, 332, 10). An antibody delivery reagent based upon this approach known as GENOMONE-CAB has been commercialised for this purpose. It claims to overcome the difficulties involved in experiments using conventional lipid-based reagents by which antibodies are introduced into cells by means of endocytosis. Similarly, in another study, a novel method for the delivery of antibodies into cells using the TAT-fused protein was reported (Lee, K. O. et al, Improved intracellular delivery of glucocerebrosidase mediated by the HIV-1 TAT protein transduction domain, Biochem Biophys Res Comms, 2005, 337, 701). This fusion protein consists of two functional domains, the protein transduction domain of HIV-1 TAT and the B domain of staphylococcal protein A (SpA), which has an ability to bind to the IgG. The TAT-SpA fusion protein was mixed with fluorescent-labelled rabbit IgG and added to cells and the internalization of antibody was analyzed using confocal microscopy and flow cytometry in living cells.
A widely reported method of antibody delivery is the use of liposomal systems. A number of relatively inexpensive lipid-based delivery systems are commercially available. For example, LIPODIN-AB™ and AB-DELIVERIN™ are antibody delivery systems that claim to deliver functional antibodies to their targets; be highly efficient in many cell lines and primary cells; be serum compatible; be suitable for all antibodies; biodegradable whilst retaining high cell viability; and are ready and easy to use. These systems are lipid-based formulations that form non-covalent complexes with antibodies through electrostatic and hydrophobic interactions. It is well-known however, that despite the claims of high cell viability, these cationic lipid and polymer-based systems are notoriously cytotoxic to cells. This would certainly be a more serious problem for any therapeutic treatment for which it is necessary to continually administer the antibody complex over a prolonged period of time.
Walter et al in Eur J Cell Biol, 1986 April; 4(2): 195-202 describe liposome-mediated delivery of antibody to a Drosophila cell line. Antibody was encapsulated together with a dye into liposomes and uptake by cells observed using light microscopy.
Lipid-based systems that rely upon endocytosis to enter the cell also encounter problems associated with inefficient release (escape) from the endosome. In 2006 Carafa M. et al. (Eur J Pharm Sci. 2006, 28, 385) presented work regarding pH sensitive vesicles that were able to escape the endosome due to a change in pH. Other work has been done in order to attempt to enhance the cytosolic delivery, for example using tertiary amine-based detergents (Asokan A, Cho M J., J Control Release. 2005, 106, 146). However this method affects the viability of cells by partially disrupting the membrane. Other trials were performed using pore-forming agents such as toxin streptolysin O (SLO) which can be used to reversibly permeabilise adherent or nonadherent cells (Walev I, et al, Proc Natl Acad Sci USA. 2001, 98, 3185). All of these methods stress the cell and may alter cellular responses giving unreliable results.
An alternative delivery system composed of a trifluoroacetylated lipopolyamine is described in Zelphati et al; Journal of Biological Chemistry; Vol. 276, No. 37, Sep. 14 2001; pp 35103-35110. This cationic lipid formulation is used to enable recombinant proteins, peptides and antibodies to enter viable cells.
A very efficient, non-toxic and non-inflammatory polymer vector for the delivery of DNA within human cells is described in Lomas, H et al. Biomimetic pH Sensitive Polymersomes for Efficient DNA Encapsulation and Delivery. Adv. Mater. Vol 19 (2007), pages 4238-4243. In addition, a combination of amphiphilic polymer with DNA is described in WO03/074090. Depending on the block lengths of the respective components of the copolymer, the interaction with DNA can be tailored to produce DNA condensates (polyplexes) or to encapsulate the DNA within a vesicle. The latter is based on the self-assembly of pH sensitive poly (2-methacryloxyethyl phosphorylcholine)-poly (2-(diisopropylamino)-ethyl methacrylate), (PMPC-PDPA) block copolymers into nanometer-sized vesicles, also known as polymersomes (Du, J., et al; pH Sensitive Vesicles based on a Biocompatible Zwitterionic Diblock Copolymer. J. Am. Chem. Soc. 127, 17982-17983 (2005)). The use of these polymer vesicles for delivering nucleic acids and rhodamine dyes is further described in Lomas et al; Faraday Discuss. 2008, 139, 143-159. None of these prior art references describe the delivery of antibodies into cells.